Our long-term goal is to elucidate the cellular functions of Pseudomonas aeruginosa required for initiating infection and needed for persistence in chronic wounds. The findings will then be used to identify effective therapeutic targets against P. aeruginosa to promote wound healing. Despite increasing evidence pointing to the inefficient elimination of opportunistic pathogens as a major reason for why chronic wounds do not heal, the molecular mechanism underlying the role of microbes in the pathogenesis of chronic wound infections remains unclear. P. aeruginosa is a key causative agent for chronic wound infections, particularly in diabetic foot ulcers. Increasing evidence suggests bacterial existence in biofilms to impede eradication by host immune response and antimicrobial treatments. Moreover, escalating emergence of resistance to existing antibiotics, lack of new antibiotic development, and rising cases of diabetes and obesity fuel the need for improved therapeutic modalities. To address this important public health issue, comprehensive identification using next-generation sequencing and chronic wound infection model in diabetic mice will be used in these specific aims: (1) Identify P. aeruginosa functions required to initiate infection and persist in chronic wound infections. To examine mutants at genome-wide scale, a recently developed transposon-sequencing (Tn-seq) technology will be employed. Tn-seq utilizes massively parallel DNA sequence analysis to track millions of bacteria carrying unique transposon insertion mutations. A library pool containing mutations in nearly all nonessential genes will be created and used to infect mouse wounds. Mutants that fail to survive in the wound environment will be identified using Tn-seq. The gene products of identified mutants may lead to potential targets for new therapeutic treatment. (2) Validate impaired infection of candidate functions identified in Aim 1. Identified functions will be reexamined in vivo using a single mutant infection. In parallel, candidate genes will be inactivated in clinical wound isolate to test whether attenuated infectivity can still be observed in clinical strain background. (3) Functionally characterize candidate genes to help understand the molecular basis underlying attenuated infectivity. Assays will test whether functions known to be involved in infection are impaired in the mutants, including biofilm formation, motility, and resistance to killing by phagocytic cells. The anticipated outcome of this project is a comprehensive identification of genes required for P. aeruginosa infection and validated potential targets for therapeutic treatment of diabetic chronic wound infections.